Fungi are an exceptional source of structurally unique and biologically active small molecules, many of which have served as inspirational leads for the development of current and evolving therapeutic agents (e.g., penicillins, echinocandins, cyclosporins, ergot alkaloids, and statins). Despite their tremendous promise, fungi have proven to be a challenging group of organisms to explore due to extensive silencing of secondary- metabolite-encoding genes. Epigenetic processes are proposed to be an important means by which fungi actively suppress the transcription of genes involved in natural product biosynthesis. We hypothesize that chemical manipulation of epigenetic targets is an effective technique for accessing structurally-unique natural products from silent biosynthetic pathways. This hypothesis is based on our group's published studies and strong preliminary data demonstrating that a chemical epigenetic approach is a practical and rationally-based method for transcriptionally activating silent biosynthetic pathways and securing their respective small- molecule natural products. Our chemical epigenetic approach to accessing fungal cryptic secondary metabolites offers several distinct advantages over current systems due to its simplicity, universal applicability, and ability to be readily incorporated into modern microbial screening programs. This NIH Roadmap initiative presents a unique opportunity for testing the central hypothesis and addressing the need of natural products researchers for an effective paradigm to access silent biosynthetic pathways. The following two specific aims will serve as the focus of our studies. Specific Aim 1 addresses the key methodological development component of RFA-RM-09-005. For this aim, we will determine the epigenetic underpinnings of secondary- metabolite-encoding gene suppression using real-time qRT-PCR and ChIP-Seq. This is expected to provide novel mechanistic insight into the role that epigenetic processes play in the transcriptional suppression of silent biosynthetic pathways, which will enable us to further refine our group's chemical epigenetic technique for the in situ mining of cryptic natural products from fungi. Specific Aim 2 serves as an experimental assessment of the broad-spectrum capacity of the chemical epigenetic methodology to yield novel metabolites from phylogenetically diverse fungi and will result in the generation of a series of structurally unique natural products that will be submitted to the Molecular Libraries Small Molecule Repository (MLSMR) for high throughput screening throughout the NIH-sponsored Molecular Libraries Probe Production Centers Network (MLPCN). Our methodology is highly innovative because it utilizes a unique epigenetic-based technique for rationally manipulating the expression of fungal silent biosynthetic pathways in situ. These results are expected to have a positive and far-reaching impact on the field of natural products by 1) providing a critical research tool that is needed to systematically explore fungal silent biosynthetic pathways and 2) affording new compounds with important biomedical/pharmaceutical applications. PUBLIC HEALTH RELEVANCE: Fungi are a highly diverse group of organisms (approximately 1.5 million species worldwide) that are responsible for producing some of the most important drugs known to humankind including antibiotics (penicillins and cyclosporins), cholesterol-lowering agents (statins), and antimigraine drugs (ergotamine). The potential for fungi to yield new therapeutic leads is tremendous, but fungi have proven difficult to work with due to their ability to block the production of natural products under laboratory culture conditions. Our group will test a new methodology based on epigenetic induction for targeting the activation of fungal genes involved in natural product biosynthesis. This method is expected to give researchers direct and immediate access to a wealth of new compounds that have the potential to aid in the study and treatment of numerous human diseases.